Damage to the nervous system, and particularly the central nervous system including brain, spinal cord, and optic nerve is believed to be irreversible, leading ultimately to the process of degeneration. Traffic accidents and sports injuries, ischemia, tumors, prolonged inflammation, cryptogenic degenerative disease and the like are among the causes of neurological diseases which occur with a high incidence among the population, and are of urgent social significance.
The irreversibility of central nerve damage is attributed to the glial environment of nerve tissue. The brain and spinal cord have the same glial environment, which will now be described using the optic nerve as an example of a central nerve.
1) First, nerve fibers undergo degeneration and gradually disappear. During the process, the myelin sheaths covering the nerves also degenerate leaving cell residues (see FIG. 1b). The myelin sheaths formed by oligodendrocytes contain substances which strongly inhibit nerve fiber regeneration and elongation(1).
2) The astrocytes proliferate and enlarge, resulting in gliosis (see FIG. 1b). More specifically, they displace the nerve fibers, occupying the primary locations and thereby physically inhibiting regeneration(2). The astrocytes forming the gliosis exhibit a morphology contrasting considerably with that of normal astrocytes, with a greater number of processes and intricately complex forms. Particularly in the case of injury, the site of damage shows numerous layers of astrocytes stacked orthogonally to the direction of extension of the nerve fibers and linked together at their processes, forming a cap-like barrier structure.
3) Processing of the oligodendrocytes and their cell residue substances such as myelin after degeneration is slower compared to other regenerating tissues such as peripheral nerve system. The main reason for this is presumably the very low degree of infiltration of peripheral immune cells such as macrophages and monocytes, resulting in delayed processing of the residue in the early stages.
The following explanations have been proposed for lack of regeneration of optic nerves.
1) As mentioned above, oligodendrocytes have a strong inhibiting effect on neural regeneration. Specifically, the molecule Nogo extracted from oligodendrocytes has been shown to be an inhibitor(1,9). Experiments with culturing systems have shown that during extension of neurites, the processes contacting with the myelin sheaths of oligodendrocytes not only stop extending, but even regress (contact inhibition). Moreover, neurites do not extend at myelinated areas, and in culturing the processes grow to avoid them.
2) Gliotic astrocytes produce various inhibiting substances, including proteoglycans such as keratin sulfate and chondroitin sulfate(3).
3) The optic nerves and the entire central nervous system are exceedingly silent even after suffering injury. The system is not under immune surveillance, and this is instead considered to be a disadvantage for regeneration. For example, the peripheral nerve system described hereunder differs from the optic nerve even in the structure of the glia, and it has been compared and studied as a regenerating system even though it is composed of the same nerve tissue. Upon injury of peripheral nerve tissue, immune cells such as peripheral macrophages rapidly infiltrate (within a few hours to a couple of days) to process the cell residue. Meanwhile, cytokines are secreted in large amounts, promoting regeneration of the nerve tissue. Such neural regeneration occurs in concert with a cascade of phenomena with one reaction leading to another, but no infiltration of macrophages is seen in the optic nerve in the early stages. This has been attributed to the suppression of macrophage activity in the central nervous system including the optic nerve(4). It appears that this suppressing function arose in order to prevent macrophage digestion of complex developed central neural nets. It has therefore been conjectured that it is the absence of the first trigger in the optic nerves that leads to degeneration instead of regeneration.
4) In order for regeneration to occur, a structural scaffolding is necessary to induce nerve fibers. However, once degeneration has occurred in the optic nerves, the route for regeneration is lost. In the optic nerves, each oligodendrocyte forms numerous myelin sheaths on the nerve fibers, and astrocytes surround the myelinated fibers and cover the unmyelinated fiber bundles (see FIG. 2, bottom). The basal membrane is present only outside of the astrocytes forming the membrana limitans glia, or in other words, the entire optic nerve may be considered to be inside a single basal membrane sheath. In the peripheral nerves, the basal membrane serves as a route for regeneration. Consequently, once the nerve fibers in the optic nerve have degenerated, the route which once existed for each of the nerve fibers is no longer present.
Yet peripheral nerves, unlike central nerves, are capable of regeneration.
Unlike central nerves, the primary cells of the peripheral nerves are Schwann cells. All of the nerve fibers, whether myelinated or unmyelinated, are covered with Schwann cells (see FIG. 2, top). Schwann cells are derived from neural crest cells, while the central glia (oligodendrocytes, astrocytes) which act inhibitorily on neural regeneration are derived from neural tubes, and therefore the sources of differentiation are different.
Peripheral nerves are believed to regenerate in the following fashion.
1) When injury such as a cut is suffered by peripheral nerves, Wallerian degeneration occurs at the peripheral end from the site of injury (see FIG. 3). The Schwann cells return to an undifferentiated state from the myelin-forming differentiated type, and are then activated to divide and proliferate, exhibiting a funicular form. In the peripheral nerves, the individual nerve fibers are independently surrounded by Schwann cells, with the outer area covered by a basal membrane (see FIG. 2). That is, each of the nerves resides within a separate basal membrane sheath. Thus even when degeneration occurs, the Schwann cells activated in the basal membrane sheath proliferate to form a funicular structure, thus providing a foothold for reconstruction of the neural network. Wallerian degeneration, therefore, is not degeneration in the strict sense but rather the first step toward regeneration.
2) Peripheral macrophages play a major role in the process of Wallerian degeneration. The macrophages infiltrate rapidly at the peripheral end of the site of injury, processing the remnants of the degenerated nerves fibers and myelin (see FIG. 3) while also secreting cytokines such as IL-1 to activate the Schwann cells. Although no definite conclusions can be drawn regarding the cause which induces filtration of macrophages, the Schwann cells themselves have been indicated as a likely candidate. In any case, it is believed that the Schwann cells activated by the macrophage signals synthesize various factors indispensable for regeneration, such as nerve growth factor which will be explained below, and guide regeneration of the nerve.
3) The myelin sheaths of Schwann cells have a low inhibitory effect. This is a major difference from the myelin of oligodendrocytes which does exhibit an inhibitory effect(5). In addition, it is known that the composition of the myelin protein of Schwann cells and oligodendrocytes differs.
Thus, even when injured, the central glia do not revert back to their differentiated state to undergo differentiation and proliferation or significantly alter their form, as do the Schwann cells of the peripheral nerves, but instead maintain their relatively differentiated phenotype. The peripheral nerves on the other hand are characterized by exhibiting a rapid, highly flexible response to injury.
Schwann cells are considered to play the following role in neural regeneration.
Schwann cells produce numerous factors and secrete them in a diffuse manner. Moreover, their own cell membrane surfaces are covered with a basal membrane, and extracellular matrix components are included therein. Cell adhesion molecules are also known to be expressed in Schwann cell membranes, and it is thought that these factors act as a whole to induce neural regeneration (see FIG. 4).
1. Secreted Factors
Schwann cells are known to produce many neurotrophic factors, among which the following are the main ones involved in regeneration: 1) the neurotrophin family, including nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4/5; 2) ciliary neurotrophic factor; 3) the FGF family, including acidic and basic fibroblast growth factor; 4) the insulin family, including insulin-like growth factor-I and II; and 5) transforming growth fator-β2 and β3.
These neurotrophins have powerful effects not only on the survival of neurons but also on neurite elongation and the like. Other factors also have neurotrophic effects and neurite elongating effects on nerves, but their action mechanisms are considered to be autocrinic since they simultaneously activate the Schwann cells themselves.
2. Extracellular Matrix Components
These include fibronectin, laminin, type IV collagen and tenascin. Based on experiments with cultured systems, it is believed that fibronectin and laminin play a supporting role in neural regeneration.
3. Cell Adhesion Molecules
A large number of cell adhesion molecules have been identified. The following description will focus on those associated with Schwann cells and neural regeneration.
1) Immunoglobulin Superfamily
NCAM (Neural Cell Adhesion Molecule) and L1 are expressed on Schwann cell membranes and play important roles as adhesion molecules during elongation of the nerve fibers as they contact with the Schwann cell scaffolding. Both are connected to the cytoskeleton and function to maintain the shape of the cell, while also accomplishing intracellular activation through inositol phosphate system and calcium channel activation. In addition, MAG (Myelin Associated Glycoprotein) is expressed between Schwann cells after nerve elongation has progressed to some degree and remyelination of the nerve fibers has begun.
2) Cadherin Superfamily
Cadherins are calcium-dependent cell adhesion molecules of which numerous types have been identified. N-cadherin is associated particularly with neural regeneration. Like NCAM and L1 of the immunoglobulin superfamily, this molecule also plays an important role during elongation of the nerve fibers as they contact with and recognize Schwann cells.
3) Integrin Superfamily
Integrins are cellular receptors for the aforementioned extracellular matrix components. They are heterodimeric molecules composed of two subunits, α and β. Like cadherins, they are also thought to link with the cytoskeleton and function directly in signal transduction between cells. Schwann cells express the α6β4 subtype which plays a role in the process of remyelination during regeneration.
Regeneration of central nerves has been attempted in the past several decades or so by numerous researchers. The following is a summary of those attempts.
1) Regeneration has been achieved by cutting portions of peripheral nerves of the hand or foot and autografting them into the central nerves. Thus, peripheral nerves presumably possess an environment which promotes regeneration of central nerves. Such studies began with research by Aguayo et al. in Canada in 1983(8).
2) As mentioned above, the central nerves themselves act to suppress neural regeneration. Reports have been published on the inhibiting effects of previously known myelin-related proteins, including Nogo factor which was described in 2000 in the journal Nature(9). According to several reports, introducing antibodies against these factors to neutralize them can induce some degree of regeneration in the central nerves.
3) Regeneration is promoted by the following two types of cell transplantation.
a) Replenishment of degenerated neurons to reconstruct the neural network. This approach employs neuronal stem cells and embryonic neurons.
b) Reconstruction by transplantation of cells capable of inducing neural regeneration (glial cells), instead of replenishing the actual neurons. It has been attempted to use peripheral nerve-derived Schwann cells, central nerve-derived glial cells or central nerve-derived ependymal cells, having neurotrophic factors introduced, olfactory nerve-derived support cells, neuronal stem cells and the like.
Both methods have advantages and disadvantages, but as yet no revolutionary method has been developed.
The present inventors have for many years been involved in development of methods of neural regeneration and reestablishment of function. We have focused particularly on a method employing Schwann cells which support the tissue structure of peripheral nerves as described in 3)-b) above. Schwann cells are present in peripheral nerves, and it has been demonstrated that they are capable of inducing regeneration not only of their own peripheral nerve tissue but also of the central nervous system, that their transplantation at sites of injury provides a foothold for regenerating fibers and leads to effective neural regeneration, and that myelin which is responsible for neural saltatory transmission as an indispensable element for normal nerve functioning can also be reconstructed by transplantation of Schwann cells. It has also been confirmed in animal experiments that transplantation of Schwann cells leads to regeneration of cut optic nerves, (central nervous system).
Nevertheless, various difficulties are encountered when the relatively simple procedure of collecting and culturing Schwann cells in animal experiments is applied to humans. For example, since Schwann cells are present in the peripheral nerves, it is necessary to extract nerve samples from the hands or feet and isolate the cells, thereby leaving damage in the donor after extraction. As an additional difficulty, the limited proliferating ability of adult-derived Schwann cells requires a greater time period for large-scale culturing. Moreover, neural crest cells, which are believed to differentiate into Schwann cells, can only be extracted from embryonic peripheral nerves.
This situation has therefore necessitated provision of a natural Schwann cell substitute which can be used for neural regeneration treatment and can be obtained in large amounts by culturing.
Neuronal stem cells have been found in portions of the adult brain, and these differentiate into the neurons, astrocytes, oligodendrocytes, etc. of the nervous system (see FIG. 5). However, only a very minute number of such stem cells are present, and craniotomy is necessary to obtain them. In addition, recent research has shown, contrary to germ layer theory, that some types of cells may be able to differentiate into completely different types (see FIG. 6). As of the filing of the present application it has been known that bone marrow stromal cells are mesenchymal stem cells or precursor cells that not only carry out a hemopoietic support function but can themselves differentiate into osteoblasts, vascular endothelial cells, skeletal muscle cells, adipocytes, smooth muscle cells and the like(10); nevertheless, no literature exists suggesting the possibility that bone marrow stromal cells might be capable of differentiating into neural crest cell-derived Schwann cells, nor has any method for such differentiation or induction been established.
In light of this situation, the present inventors have attempted experimentation and research on differentiation and induction to Schwann cells using bone marrow stromal cells instead of neural crest cells that are so difficult to obtain, as mentioned above. Bone marrow stromal cells are easy to extract by bone marrow puncture on an outpatient basis and have high proliferation potency, and thus allow large-scale culturing in a relatively short period of time.